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Stephen Rothman Abstract Unlike most substances that cells manufacture, proteins are not produced and broken down by a common series of chemical reactions, but by completely different in

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Open Access

R E S E A R C H

Research

How is the balance between protein synthesis and degradation achieved?

Stephen Rothman

Abstract

Unlike most substances that cells manufacture, proteins are not produced and broken down by a common series of chemical reactions, but by completely different (independent and disconnected) mechanisms that possess no intrinsic means of making the rates of the two processes equal and attaining steady state concentrations Balance between them is achieved extrinsically and is often imagined today to be the result of the actions of chemical feedback agents But however instantiated, chemical feedback or any similar

mechanism can only rectify induced imbalances in a system previously balanced by other means Those "other means" necessarily involve reversible mass action or

equilibrium-based interactions between native and altered forms of protein molecules somewhere in time and space between their synthesis and degradation

Introduction

While developing successful all-encompassing or general models to account for life's prop-erties is the hope of much scientific research in biology, life's varied and complex nature at times seems to preclude easy generalization Protein metabolism, the events that make and degrade proteins as well as the mechanisms that regulate the rates of these processes, is a case in point Not only is each protein, for instance the many thousands of different kinds manufactured by eukaryotic cells, structurally and functionally unique, so is the path, vari-ety, variability, and duration of their life history After synthesis, some undergo major physi-cal and chemiphysi-cal changes for reasons as varied as the changes themselves, while others seem

to remain essentially unchanged In the process of change they may be added to or reduced

in size, or they may be modified time and again as they perform a continuing function In addition, some are destroyed almost as rapidly as they are made, while others last a lifetime,

or as in growing bacterial cultures are only broken down when cell division ceases or as with the enucleate red blood cell when the cells that contain them are destroyed or as with the apoprotein of the retina in the order in which they are made In yet other cases, for instance

as part of an immune response or during development, they are only expressed for brief periods of time under very particular circumstances The complexities of the life history of proteins are enormous, as or more complex than the structure of these most complicated of molecules, and in some respects matches, perhaps unsurprisingly the complexity of life itself

Given such facts, despite the enormous experimental knowledge base about the produc-tion and destrucproduc-tion of proteins, it is not surprising that the important quesproduc-tion about pro-tein metabolism posed in this paper's title, "How is the balance between propro-tein synthesis

* Correspondence:

stephen.rothman@ucsf.edu

1 University of California, San

Francisco, San Francisco, CA

94143, USA

Full list of author information is

available at the end of the article

and degradation achieved?" has not only not been answered, to the best of my knowledge

it has never been explicitly asked This even though in the fullness of time balance

between the rates of manufacture and destruction, between what is made and what is

broken down occurs and is quantitative whatever the protein, however and wherever

degradation takes place, and even though most proteins in eukaryotes, in both the

cellu-lar and extracellucellu-lar compartments of metazoans, as well as in non-growing bacterial

cultures, are present at stable and reproducible concentrations for a given physiological

steady state, signifying balance between their rates of synthesis and breakdown

Further-more, when changes in concentration occur, due either to altered physiological

circum-stances or the presence of disease, a new steady state concentration is usually sought and

found

But the absence of discussion should not be taken to mean an absence of opinion

Thomas Kuhn in describing the nature of the scientific paradigm argued that there are

really no open questions, or at least no open questions of significance in scientific

disci-plines Whether supported by evidence and reason or merely expressions of bias, the

paradigm leaves no question unanswered, even if only implicitly In this regard, things

are particularly difficult for protein metabolism Because proteins are central to virtually

every area of biology, from molecular biology, to biophysics, to structural biology, to

microbiology, to biochemistry, to cell biology, to immunology, to pathology, to

physiol-ogy and systems biolphysiol-ogy, there are often different, non-commuting disciplinary

perspec-tives In this regard, in what follows we will consider lysosomal degradation, feedback

regulation, and the equilibration of native and altered proteins as potential answers to

the question posed in the article's title

In any event, taken together such circumstances are not only ripe for strong differ-ences of opinion, but make attempts to generalize about how balance is achieved

daunt-ing And yet, science cannot simply demur and decide that the question not only can't be

answered, it shouldn't be asked, or that asking it is a pointless or fruitless exercise It is

duty bound to seek broad explanatory rules however seemingly complex and varied the

phenomena The analysis that follows is based on fidelity to this belief, with appreciation

for the difficulty of the task at hand and awe at life's still unexposed mysteries

Background

With some exceptions such as growing bacterial cultures, even a small persistent

imbal-ance between the rates of synthesis and degradation of proteins is inimical to cellular and

organismal life1 Over the past half-century we have learned a great deal about how

pro-teins are manufactured and degraded, the rates at which they turn over, and how these

processes are regulated However, little attention has been paid to how balance, or parity,

between the two is achieved

For most substances that cells manufacture their rate of formation, or anabolism, and the rate of their breakdown or transformation, or catabolism, are balanced by mass

action, expressed in common or related chemical reactions and intermediate states (e.g.,

A + B < > C < > D + E) Things are entirely different for proteins Most importantly,

the mechanisms responsible for their manufacture and breakdown are not part of a

com-mon chemical process, but are completely independent of each other both chemically

and physically In addition, and also unlike other molecules, the rates at which these

pro-cesses occur is not determined by the rate at which the chemical bonds that form the

substances are made or broken, but by external factors, for example, the amount of

mRNA for synthesis or the rate of ubiquitination for degradation [1,2]

Finally, both synthesis and degradation are irreversible processes in that they are unre-sponsive to mass action effects of their end products (proteins and amino acids

respec-tively) on their rates For example, if we take a protein and break it down to its

substituent amino acids, not even a small amount will reassemble spontaneously Protein

synthesis is the most expensive biosynthetic process known to us, and reconstruction in

the absence of a great deal of free energy is extremely unlikely, but even if the energy

were available, without a means of generating the appropriate sequence of amino acid

subunits, as is done by mRNA during synthesis, the authentic peptide chain simply

can-not be reconstituted

Nor is a mass action effect of a protein on its own rate of synthesis any more likely

Once manufactured, the new protein is released from the synthetic machinery of the

ribosome into the cytosol or other cellular compartment As such, it cannot affect

upstream events on the ribosome by mass action Indeed, there are no upstream events

to affect Ribosomes are assembly lines for the construction of single peptide chains [3,4].

As the nascent chain moves through the ribosomal machinery, no other chains are being

produced behind it on the same ribosome The process is discontinuous, and after a new

protein is discharged, the ribosome becomes inactive Its two major subunits dissociate

until a new mRNA molecule comes along to start the process over again, in all likelihood

for a different protein

Lysosomal degradation

According to one line of current thinking, there are two general mechanisms for the

deg-radation of proteins in eukaryotic cells, one for cytosolic and nuclear proteins, and

another for proteins that are contained in or are part of large intracellular structures

(excepting the nucleus), such as various membrane-enclosed vesicles and organelles For

cytosolic and nuclear proteins, breakdown occurs within proteasomes, small

freestand-ing pore-like aggregates of degradative enzymes and regulatory proteins found in the

cytosol and nucleoplasm [5-10] It is thought that dysfunctional structural changes occur

to protein molecules over time due to random environmental causes, or as a result of

being defective initially, and that as a consequence certain exposed regions on the altered

molecules serve as the predicate for their degradation For at least some, a small protein,

ubiquitin, affixes to particular imperfections and marks them for destruction [11-16] In

the other degradative system, entire anatomical structures enter small

membrane-enclosed sacs known as lysosomes as the result of membrane fusion [17-19] Subsequent

to fusion, lysosomal enzymes disassemble and degrade the structure and its contents,

including its proteins

While the proteasome system appears capable of achieving metabolic balance (see below), the lysosomal system does not Though lysosomes may disassemble and degrade

foreign bodies and their contained chemicals [20], or be responsible for autophagic

responses to cellular pathology and aging [21], they seem ill suited to balance the

ongo-ing manufacture and degradation of endogenous proteins There are two reasons

First, by necessity the rate-limiting step in lysosomal degradation is membrane fusion

Otherwise, the fused objects would continuously accumulate in the cell in anticipation of

processing This does not normally occur, and cellular life could not be sustained if it did

As a consequence of fusion being the rate-limiting step, all of the proteins in a given

structure would have to be degraded and, to achieve balance, synthesized at a single

common rate, the rate of fusion This is not the case Different proteins are made and

degraded, turned over, at distinctive and often quite disparate rates even when present in

the same structure

The second reason is even more compelling To achieve balance cells would actually

have to know the rate of fusion, as well as the protein contents of the fused objects, the

mathematical product of the two, and have the means to transmit this information to the

synthetic machinery As we understand biological cells, such tasks lie beyond their

capa-bilities They have no more knowledge of what they are doing than clouds or rivers In

any event, in what follows I only consider proteins that are broken down in proteasomes,

even though the general requirements for producing metabolic balance apply whatever

mechanism is employed2 In addition, I only take into account proteins that are present

at stable values for particular physiological steady states

The effect of feedback

If the mechanisms that determine the rate at which a protein is made and those that

determine the rate at which it is broken down possess no intrinsic means of making the

two equal, then balance between them requires a mechanism that is extrinsic to these

processes As said, this is usually, though not uniformly, imagined today in terms of

chemical feedback Chemical agents or signals, acting separately or together,

synergisti-cally or antagonistisynergisti-cally, on one process or both, feedback on various steps in synthetic

and degradative pathways adjusting their rates to achieve metabolic balance (figures 1

and 2)[1,2]

For synthesis, the feedback agents alter the production of mRNA from its DNA tem-plate (transcription)[1,2,22-25], as well as its availability and effectiveness subsequently

(post-transcriptionally)(figure 1)[26-30] For degradation they in the main act on the

events that immediately precede breakdown, that is, on choosing or preparing molecules

for degradation (figure 2)[1,2,31-38]

And yet unlike chemical reactions where mass action produces balance between pro-duction and breakdown automatically as the reaction seeks a steady- or equilibrium

state, for a negative feedback agent or other extrinsic mechanism to achieve balance

requires something quite different and as it happens quite unlikely That is, the

mathe-matical product of its concentration and the avidity with which it binds to relevant sites

must combine to alter the rate of manufacture or breakdown of the substance by just the

right amount so that by fortunate circumstance it equals the rate of the countervailing

process For example, if the feedback agent were the end product of an enzyme-catalyzed

chemical reaction, its concentration and the avidity with which it binds to the catalyst

would have to combine to produce a change in the catalyst's effectiveness that by chance

would alter the reaction's rate to the same degree that it would have been altered if the

end product had acted by mass action For the transcriptional regulation of protein

syn-thesis, this would require that a particular concentration of a feedback agent bind to a

regulatory protein with an avidity that produces a concentration of the resultant complex

that binds to DNA with just the right affinity so that in repeated acts of association and

dissociation, transcription is turned on and off at a frequency that produces an amount

of mRNA that yields a rate of protein synthesis equal to that of degradation [1] Such a

concatenation of events seems implausible

Given the unlikelihood of these and analogous circumstances, chemical feedback,

whatever its incarnation, can only rectify induced imbalances in a system already

bal-anced by other means It cannot establish that balance in the first place For example, if

an increase in the concentration of a protein occurs due to an elevation in its rate of

syn-thesis, feedback can produce a proportional increase in the rate of degradation, forcing a

return to a prior concentration and state of balance If however the two rates were

unequal initially, increasing them in proportion to each other would not make them

equal In this case, if a = b, then 2a = 2b, but if a > b, then 2a would remain greater than

2b (2a > 2b)3

Protein turnover

To establish, as opposed to re-establish balance between synthesis and degradation, a

different sort of mechanism is needed An important clue to that mechanism was

discov-ered many years ago in studies on protein turnover the renewal and replacement of

proteins [39-45] In this large body of work, a protein's rate of turnover was commonly

estimated by producing a dislocation from a prior steady state concentration by

increas-ing or decreasincreas-ing its rate of synthesis or degradation artificially and then passively

Figure 1 The feedback regulation of protein synthesis Shown are events of protein synthesis that are

af-fected either directly or indirectly by feedback agents (italics)(see The effect of feedback).

DNA RNA transcript

mRNA

transcription

processing

nuclear membrane

transport and localization

initiation elongation release

RIBOSOME

protein

active protein

activation

promoter

RNA polymerase

RNA DNA

observing as a new steady state was approached, or by following the disappearance of

radio-labeled proteins from previously labeled cells and tissues, or by measuring the

incorporation of radioactive amino acids into cellular protein The mathematical

func-tions that described these phenomena exposed the kinetic nature of the mechanisms

that produce balance, though they did not disclose their details

As expected, given its irreversible character, synthesis was a zero-order or linear pro-cess But degradation, also understood to be irreversible, was not Its kinetics was

first-order or exponential, and suggested a reversible or equilibrium-dependent event Given

what I said about the irreversibility of degradative processes, this is deeply contradictory

We would expect degradation to be a zero-order process, just like synthesis And yet this

said, what was observed was entirely predictable If both synthesis and degradation were

zero-order or linear processes, life could not be sustained Since linear functions do not

converge, any difference in the rates of synthesis and degradation would continue ad

infinitum; there could be no equilibration, no steady state Of course, degradation

occur-ring more rapidly than synthesis is a non sequitur since the cell would be void of the

pro-tein forever, but assuming that the rate of synthesis is greater, the concentration of the

substance would rise monotonically and ceaselessly over time

The contradiction that this irreversible chemical process is of the first order can only

be resolved if the first order kinetics are not attributable to degradation itself, but to a

foregoing process that sets its rate This foregoing process must be reversible, in other

words equilibrium-based mass action That this is so is indicated variously by the

chemi-Figure 2 The feedback regulation of protein degradation Shown are events of proteasomal degradation

(for an ubiquitination system) that are affected either directly or indirectly by feedback agents (italics)(see The effect of feedback).

ubiquitinated protein active protein

polyubiquitination

peptides

amino acids

ubiquitin + E 1 activating enzyme

E 2 E 3 complexing ligase

polymerization

unfolding and presentation (cap)

proteolysis (cylinder) PROTEASOME

cytoplasmic proteases

protein

cal or reaction-based nature of turnover, the first-order isotope kinetics seen at the

steady state (when production and degradation are equal), and the tracer kinetics of

iso-tope incorporation studies4 As a consequence, degradation need not impossibly be both

reversible (first-order) and irreversible (zero-order) at one and the same time It is an

irreversible process whose rate is regulated by a preceding reversible event

Equilibration

This reflects a primary causal discernment As a general matter, chemical events are

bal-anced by mass action among their constituents, and despite the unique circumstances of

protein synthesis and degradation, physical law does not allow an exception to this rule;

it provides no other means of achieving balance The question then is not whether this

occurs, but how and where? If balance is not achieved within the synthetic and

degrada-tive reaction sequences in their own right, and it is not, then it must take place external

to them That is, the mass action event must occur somewhere in time and space

between the manufacture of proteins on ribosomes and their breakdown in

protea-somes, in other words in the solvent phases of the cell that contain both of these

structures5

Among which molecules would this mass action effect occur? Based on the under-standing that each protein turns over at a unique rate, equilibration must be between the

"native" protein physiologically capable or mature forms of the molecule and modified

or altered forms of the same protein that are predisposed to degradation and that set its

rate The equilibrium constant between the two forms reflects their ratio in the cell at

the steady state In this way, the rates of the irreversible and independent mechanisms of

synthesis and degradation are joined and balanced both in the first instance and

subse-quently

Evidence

While the existence of such an equilibration, however it is executed, is as true as the

assumption that the synthesis and degradation of proteins are equal at the steady state,

as with any theoretical conclusion experimental validation is important As such, we

should ask whether there is evidence for the predictions of the inference and where there

is none, is it susceptible to experimental verification?

Regarding the evidence on hand, three important predictions have not only been vali-dated, but are well established First, research, most importantly on the ubiquitin system

[11-16] as well as on defective (DRiPS) proteins [46-51], has shown that many proteins

are altered after their synthesis in ways that predispose them to degradation Second,

turnover studies demonstrate that the rate of degradation is indirectly driven by the

con-centration of the protein substrate And finally, also from turnover studies, the first order

kinetics of degradation, combined with the irreversible nature of the degradative

pro-cess, is proof of equilibration prior to degradation

Together these facts provide substantial validation for the proposal protein mole-cules exist that are predisposed to degradation, the rate of degradation is driven by

con-centration, and equilibration occurs between different forms of the protein prior to its

degradation All that is missing, and this is not to minimize it, is evidence showing which

molecules that are predisposed to degradation are reversibly related to a native form

That is, which particular molecular variants in the chain of events from synthesis to

deg-radation equilibrate? The challenge in obtaining this evidence is not methodological

[equilibration can be studied in many ways, in cell free systems, as well as in vivo (e.g., by

inhibiting degradation)], but in determining which molecules are involved And at least

initially, finding the relevant molecules may be more a matter of trial and error, than a

priori determination

Equilibrating pools of protein

Beginning with the pioneering work of Wheatley in the 1980s [46-51], we learned that

protein synthesis is error-prone On average 30% (in some cases as high as 90%) of new

protein is defective and improperly folded Even for proteins transferred into the

endo-plasmic reticulum (ER) as they are synthesized, the defective molecules are transported

back into the cytosol to be degraded by the ubiquitin/proteasome system This transfer is

not an oddity, but evidence of a more general fact

If we exclude lysosomal degradation as a means of achieving balance, absent some yet undiscovered mechanism, all proteins, including those embedded in membranes and

contained within intracellular membrane-enclosed structures, must have access to the

proteasome system in the cytoplasm or nucleus to be degraded6 For this to occur, they

must have cytosolic (or nuclear) compartments But more than that, for turnover to

apply to the whole cellular pool, as it ultimately must, this compartment must be in

equi-librium with the remainder of its cellular contents wherever they are located In

accor-dance with this conclusion, Rock, et al found that inhibition of proteasome function

produces the almost complete inhibition of protein degradation [52]

Conclusion

Some of the description given above of the various processes involved in protein

metab-olism, of synthesis, degradation and their regulation, has of necessity been abbreviated

and in many areas lacks details that are no doubt important to specialists This is for

rea-sons of space the details of fact and evidence in the many fields involved is truly

enor-mous and clarity the belief that extraneous detail would obscure otherwise relatively

straightforward concepts Whatever problems these deficiencies of detail and subtlety

introduce, they do not make the presentation less salutary, the issues less cogent or the

conclusions less clear The conclusions, whether about lysosomal degradation, feedback

regulation, or most importantly about equilibration, tell us, independent of mechanistic

details, what must occur and what cannot occur as a matter of logic and our

understand-ing of physical and chemical kinetics

The principal conclusion to be drawn from this analysis is that through the agency of mass action and the conservation of mass, the equilibration of complementary forms of

the same protein molecule sets and balances its rate of synthesis and degradation The

difference between proteins and most other bioorganic molecules in achieving this

bal-ance is that for proteins the mass action effect occurs, in time and space, between their

production and breakdown, not as part of it As explained, physical law requires a means

of equilibration, and the separate nature of synthetic and degradative mechanisms by

necessity place this event in the solvent phases of the cell that contain ribosomes and

proteasomes

Though turnover studies in the past and the more recent discovery of the ubiquitin pathway provide important evidence for the presence of this equilibration, the current

analysis makes it clear that equilibration cannot be part of degradation per se, and that

the ubiquitin or any foregoing pathway must include an equilibrating element If

atti-tudes are not too hardened, the assessment presented in this article can serve as a helpful

starting point for further exploration of this important, but long ignored subject

Appendices

Appendix 1

A small uncompensated difference, say 1%/day, between the rates of synthesis and

degra-dation of a protein in a cell with a longevity of a year, say a liver cell, would produce

enor-mous and unsustainable changes in its concentration during the cell's lifetime This said,

whether in cells or extracellular fluids, most proteins are found at characteristic steady

state or stable values for specified functional states, even in non-dividing bacterial

cul-tures For example, in a large-scale multivariate study in E coli, balance between protein

production and destruction was found for many proteins under all sorts of conditions

[53,54]

Appendix 2

In this light, inhibition of proteasome function produces the almost complete inhibition

of protein degradation [52]

Appendix 3

For all equilibrium-based physical states and chemical reactions, proteins included, the

relationship between the rate constants for synthetic and degradative reactions are

pro-portional or multiplicative (Ks/Kd) [most simply, dPx/dt = (Ks/Kd) Px(t), where P is the

amount of protein x] In feedback and other similar mechanisms, the constants are

related in a subtractive fashion [for kinetics of the same order Ks - Kd or for different

orders Ks - f (Kd)], and as such balance can only be achieved at a particular invariant

concentration (when the difference is zero)

Appendix 4

These facts eliminate the possibility of an irreversible first-order process analogous to

isotopic decay

Appendix 5

If a protein's concentration were to directly drive the rate of degradation to match that of

synthesis without the intercession of a reversible mass action process, the kinetics of

degradation would be zero-order, and as explained this is not the case Also,

concentra-tion is a measure of the difference between the rates of formaconcentra-tion and breakdown, not

their separate and distinctive magnitudes, and as such cannot be used to establish

bal-ance between them

Appendix 6

Some degradative enzymes are found in the mitochondrion and it is possible that at least

some mitochondrial proteins are degraded locally

Competing interests

The author declares that he has no competing interests.

Author Details

University of California, San Francisco, San Francisco, CA 94143, USA

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Received: 15 June 2010 Accepted: 23 June 2010

Published: 23 June 2010

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© 2010 Rothman; licensee BioMed Central Ltd

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